Modified arsenite oxidase and a biosensor for detecting arsenite

ABSTRACT

The present invention provides an arsenite oxidase enzyme modified to prevent translocation by modification of a translocation signal sequence. A microorganism modified to express the heterologous arsenite oxidase enzyme is also provided by the invention, together with a device for detecting the presence of arsenite in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/352,437, filed on Apr. 17, 2014, which is a National StageApplication of PCT No. PCT/GB2012/052609, filed on Oct. 19, 2012.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, was submitted Apr. 17, 2014,in U.S. application Ser. No. 14/352,437 (National Stage ofPCT/GB2012/052609). Said ASCII copy, created on Apr. 15, 2014, is namedSequence Listing for GILJEN 3.3-012 (E) ST25.5XT, is 16,442 bytes insize, and was transferred to the present application.

FIELD OF THE INVENTION

The present invention relates to modified enzymes for detecting thepresence of arsenic derivatives, in particular arsenite, and methods forexpressing such enzymes in heterologous organisms. The invention is alsodirected to devices for detecting arsenic, and derivatives thereof, in asample.

BACKGROUND OF THE INVENTION

It is said that the third major challenge for a sustainable future(together with food security and energy) will be making the best use oflimited supplies of pure water for both agricultural use and humanconsumption, and the remediation of marginal and contaminated water willbe essential in achieving this. Already groundwater contamination,resulting from either natural geochemical processes or industrialactivities such as mining, is a major problem in many countries.

Arsenic (As) is a groundwater contaminant that is ubiquitous in theenvironment and the two soluble forms, arsenite (As^(III)) and arsenate(As^(v)), are toxic. Anthropogenic activity has resulted in widespreadcontamination of both soluble forms but As^(III) is prevalent in anoxicenvironments, including most sources of drinking water. Countriesaffected include India, Bangladesh, Vietnam, USA, Germany, France,Hungary, Australia, Argentina, Mexico, Canada.

An important aspect of remediation is assessment and monitoring, andwhilst laboratory methods exist that demonstrate high specificity andsensitivity (e.g. ICP-MS or HPLC) it is also possible, and indeeddesirable, to measure analytes such as arsenite in the field usingsensors. Ideally, the sensors should be low-cost, disposable and able tobe readily adapted to multiple analytes that are commonly found togetherin contaminated water.

Many As filed test kits are commercially available (e.g. from IndustrialSystems, Inc. Hydrodyne) but these only detect total As, rather than themost toxic form, As^(III), which is dominant in anoxic drinking waters.Moreover, because As remediation preferentially removes As^(v) (e.g. bybinding to iron hydroxide) and requires the pre-oxidation of As^(III),it is crucial to determine whether any As^(III) remains in the water.The chemically based arsenic field kits rely on a colorimetric methodwhich reduces the As^(III) and As^(v) to the gas arsine which reactswith the mercuric bromide test strips. These kits require the trainingof personnel, are expensive (e.g. Arsenic, Quick II Hydrodyne kitUS$4.24 per test and Ultra Low Quick II, Industrial Test Systems, Inc.US$6 per test) and have low sensitivity and reproducibility.

Whole cell biosensors have been developed for the detection of As^(III)by a number of groups (e.g. Stocker et al. (2003) Environ. Sci. Technol.37, 4743-4750). These methods are all based on colorimetric assays thatsometimes require the use of a luminometer. They all use the regulatorymechanism of the Escherichia coli arsenic resistance system whichdetects both As^(III) and antimonite (Sb^(III)). The regulatory gene inthis system, arsR, is fused to a reporter gene (e.g. luciferase gene,luxB) that when expressed after induction with As^(III) produces avisible signal (e.g. fluorescence).

There are many problems with whole cell based As^(III) biosensors,including: 1) the system is too complex and because of this has a slowresponse time (i.e. As^(III) must enter cells followed by induction ofregulator-reporter gene protein—this can take up to 24 hours for aresponse); 2) lack of specificity as the system does not discriminatebetween A^(III) and Sb^(III); 3) incubation temperatures of 30° C. areoften required; 4) colorimetric assay often requires use of aluminometer, which is not feasible at most field sites; and 5) use ofgenetically modified organisms always presents an additional problem. Nowhole cell biosensors for the detection of As^(III) are commerciallyavailable.

A biosensor for As^(III) has been developed using molybdenum-containingarsenite oxidase (known as “Aio” and also previously known as Aro andAso; see Lett et al., Unified Nomenclature for Genes Involved inProkaryotic Aerobic Arsenite Oxidation; J. Bacteriology, 4 Nov. 2011;p.20′7-208) which is a member of the DMSO reductase family, preparedfrom chemolithoautotrophic Alphaproteobacterium Rhizobium sp. strainNT-26 (Santini et al. (2000) Appl. Environ. Microbiol. 66, 92-97).

As^(III) oxidase catalyses the oxidation of As^(III) to As^(v) and thesuitability of this native enzyme for use as a biosensor has been testedand shown to detect down to 1 ppb As^(III), which is 10 times lower thanthe recommended WHO MCL (maximum contaminant level) of As in drinkingwater. Furthermore the native enzyme shows specificity for As^(III)(Male et al. (2007) Anal. Chem. 79, 7831-7837). The biosensor comprisesthe enzyme directly linked to the surface of a mulitwalled carbonnanotube-modified electrode, in which electron transfer proceedsdirectly from enzyme to electrode. The authors noted, however, thatcertain commonly-used electrode materials, in particular glassy carbon(GC), were not suitable for direct electron transfer in thisconfiguration.

Heterologous expression of molybdenum-containing enzymes, especiallymembers of the DMSO reductase family, is notoriously difficult.Recently, the dissimilatory arsenate reductase from Shewanella sp. str.ANA-3 was expressed in Escherichia coli but comparisons with the nativeenzyme were not made (Malasarn et al. (2008) J. Bacteriol. 190,135-142). Expression was optimal when E. coli was grown anaerobicallywith DMSO although other electron acceptors for anaerobic growth werenot tested and neither were different strains.

Since the entire native Aio is poorly expressed in a heterologousexpression system, such as E. coli, use of this enzyme in routinedetection of As^(III) is not commercially viable.

As such, there is a need for improved methods and sensors for cheap andeffective detection of As^(III) in liquids such as drinking-water,waste-water and biological samples.

BRIEF SUMMARY OF THE INVENTION

According to a first embodiment, the present invention provides anarsenite oxidase enzyme modified to prevent translocation to theperiplasm, wherein the enzyme comprises the native AioA subunit fromNT-26 and the native AioB subunit from NT-26, wherein a portion of thenative AioB subunit corresponding to the translocation signal sequence,or a functional fragment thereof, is modified.

According to a second embodiment, the present invention is directed tothe use of a modified As^(III) oxidase according to the first aspect ofthe invention as a biosensor to detect the presence of As^(III) in asample.

According to a third embodiment, the present invention provides amicroorganism, modified to express a heterologous As^(III) oxidaseaccording to the first aspect of the invention.

According to a fourth embodiment, the present invention provides amethod of producing recombinant As^(III) oxidase, comprising expressingthe modified enzyme heterologously according to the first aspect of theinvention in a microorganism.

According to a fifth embodiment, the present invention provides a devicefor detecting the presence of As^(III) in a sample, comprising at leastone electrode, an arsenite oxidase enzyme and a redox mediator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the micro-structured surface of a device according to theinvention;

FIG. 2 is an AsIII dose-response curve showing the comparable results ofthe sensor of the invention over two consecutive days;

FIG. 3 is a comparison of AsIII oxidase activities in total cellextracts of E. coli strains DH5α_0 and JM109λpir grown with differentelectron acceptors;

FIG. 4 shows a SDS-Polyacrylamide gel (12%) of purified recombinantNT-26 AsIII oxidase (M: Molecular weight marker: phosphorylase b (94kDa), albumin (67 kDa), oval albumin (43 kDa), carbonic anhydrase (30kDa), trypsin inhibitor (20 kDa), β-lactalbumin (14 kDa) (GE Healthcare)1: Purified recombinant AioBA, two subunits AioA (-94 kDa) AioB withN-terminal His-tag (-19 kDa));

FIG. 5 (a-j) show cyclic voltammogram measurements and chronoamperometry for a device according to the invention including ferrocenemethanol as the mediator;

FIG. 6 (a-d) shows show cyclic voltammogram measurements for a deviceaccording to the invention including Tris(2,2′-bipyridine)dichlororuthenium(II) as the redox mediator;

FIGS. 7a, 7b and 7c show cyclic voltammogram and chrono amperometrymeasurements for a device according to the invention including feccocenecarboxylic acid as the mediator;

FIG. 8(a-f) show cyclic voltammograms measurements for a deviceaccording to the invention including 2,6-dichlorophenolindophenol as theredox mediator; and

FIG. 9 shows cyclic voltammograms of TTF as the redox mediator.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed a modified version of the nativeAs^(III) oxidase from Rhizobium sp. NT-26 (GenBank Accession NumberAY345225) which can be successfully expressed in heterologous expressionsystems such as E. coli. This has been achieved by modifying atranslocation signal sequence present in the native enzyme, or afunctional fragment thereof, to prevent translocation.

The native As^(III) oxidase consists of two heterologous subunits: AioBis the small subunit largely composed of beta sheets and AioA is thelarge subunit largely composed of alpha-helices (Santini & vanden Hoven(2004) J. Bacteriology. 186(6):1614-1619). The polypeptide sequence ofwild-type AioB from Rhizobium sp. NT-26 is shown in SEQ ID No. 1 and SEQID No. 5 shows the corresponding nucleotide sequence. The AioB subunitcomprises a Tat leader sequence (also referred to herein interchangeablyas a Tat translocation signal sequence) which corresponds to the first25 amino acids of SEQ ID No 1. This translocation signal sequence isshown as SEQ ID No. 2 and SEQ ID No. 6 shows the nucleotide sequence ofthe portion of the aioB gene encoding the translocation signal sequence.The signal sequence directs the transport of the protein to theperiplasm using the Twin Arginine Translocation (Tat) pathway.

The present inventors have modified the native As^(III) oxidase bymodifying the translocation signal sequence in the AioB subunit. As aresult of the modification, the modified enzyme is not exported from thehost cytoplasm, and as a result can be expressed in large,commercially-viable quantities.

The translocation signal sequence can be modified by various methodswhich will be apparent to a person skilled in the art, includingframe-shift mutations, substitution mutations or deletion. Anymodification that results in loss of function of the nativetranslocation signal sequence is within the scope of the invention,however deletion of the translocation signal sequence or a functionalfragment thereof, is preferred.

Accordingly, a first aspect of the invention provides an As^(III)oxidase modified to prevent translocation by modification of atranslocation signal sequence, or a functional fragment thereof.

The modified As^(III) oxidase comprises two subunits. The first subunitcorresponds to the native AioA subunit from NT-26, or a variant,derivative or homologue thereof. The second subunit corresponds to thenative AioB subunit from NT-26, or a variant, derivative or homologuethereof; however a portion of the native AioB subunit which correspondsto the translocation signal sequence, or a functional fragment of thetranslocation signal sequence, is modified in the enzyme of theinvention. Preferably, the portion of the native AioB subunit whichcorresponds to the translocation signal sequence, or a functionalfragment thereof, is modified by deletion.

In one embodiment, at least a portion of the aioB gene which encodes thetranslocation signal sequence, or a portion thereof encoding afunctional fragment of the translocation signal sequence, is modified,preferably by deletion.

The portion of the aioB gene sequence encoding the translocation signalsequence is identified herein as SEQ ID NO. 6. Either the completesequence identified as SEQ ID NO. 6 or a homologue of this sequenceencoding a functional fragment of the translocation signal sequence maybe modified according to the invention. As a result of the modificationto the nucleotide sequence, the modified enzyme of the invention doesnot comprise the amino acid sequence identified herein as SEQ ID No. 2,or does not comprise a portion thereof that is required for a functionaltranslocation signal sequence.

As used herein, the term ‘functional fragment’ means that the portion ofthe nucleotide sequence that is modified (e.g. by deletion) encodes apolypeptide having Tat translocation signal sequence activity,preferably having at least the same activity of the polypeptide shown asSEQ ID NO: 2. As a result of the modification, the recombinant enzyme ofthe invention lacks such Tat translocation signal sequence activity.

As used herein, the term “homologue” refers to a nucleotide sequencethat encodes a polypeptide which has Tat translocation signal sequenceactivity. With respect to sequence similarity, preferably there is atleast 60%, more preferably at least 70%, more preferably at least 75%,more preferably at least 85%, more preferably at least 90% sequencesimilarity between SEQ ID NO. 6 and the sequence of the native aioB genethat is modified according to the invention. More preferably there is atleast 95%, more preferably at least 98%, sequence similarity. Theseterms also encompass allelic variations of the sequences.

In another embodiment, the portion of the aioB gene that is modifiedaccording to the invention comprises or consists of the sequenceidentified herein as SEQ ID NO. 6.

SEQ ID No. 3 corresponds to the amino acid sequence of the AioB subunitexcluding the entire native leader region. SEQ ID No. 7 shows thecorresponding nucleotide sequence. If the entire AioB leader region isdeleted, then the AioB subunit of the modified enzyme of the inventionwill correspond to SEQ ID No. 3. However, it is within the scope of theinvention for a portion of the leader region within the AioB subunit toremain unmodified, provided that the portion of the leader region thatis modified renders the remaining portion non-functional.

SEQ ID No. 4 shows the amino acid sequence of the AioA subunit and SEQID No. 8 shows the corresponding nucleotide sequence.

It is within the scope of the invention for the modified As^(III)oxidase to comprise homologues, variants or derivatives of SEQ ID Nos. 3and 4.

The terms “variant”, “homologue”, “derivative” or “fragment” as usedherein include any substitution, variation, modification, replacement,deletion or addition of one (or more) amino acid from or to a sequence.The variant may have a deletion, insertion or substitution variation.The variation may produce a silent change and a functionally equivalentpolypeptide, or may result in improved catalytic function or othercharacteristics of the resulting enzyme. Deliberate amino acidsubstitutions may be made on the basis of similar physio-chemicalproperties such as size, charge and hydrophobicity in order to alter thecatalytic function or other properties or characteristics of the enzyme.

Unless the context admits otherwise, references to “AioA” and “AioB”include references to such variants, homologues and derivatives of thenative subunits. The term “homologue” covers identity with respect tostructure and/or function and is used to refer to peptides that sharelevels of sequence identity or similarity to SEQ ID Nos. 3 and 4 andretain at least the functionality of the native amino acid sequences.The variants may result in improvements in the catalytic activity orother properties of the resulting enzyme. These terms also encompasspolypeptides derived from amino acids which are allelic variations ofthe aioA and/or aioB nucleic acid sequences (SEQ ID Nos. 7 and 8).

Levels of identity or similarity between amino acid sequences can becalculated using known methods. Publicly available computer basedmethods include BLASTP, BLASTN and FASTA (Atschul et al., Nucleic AcidsRes., 25: 3389-3402 (1997)), the BLASTX program available from NCBI, andthe GAP program from Genetics Computer Group, Madison Wis.

It is preferable if there is at least 60% sequence identity orsimilarity to the specified peptides of SEQ ID Nos. 3 and 4, preferably70%, more preferably 80% and most preferably greater than 90%, e.g. atleast 95% to the sequences of SEQ ID Nos. 3 and 4. The removal of theTat leader sequence or a functional fragment thereof to prevent exportto the periplasm is a known technique, and has been used previously inthe production of heterodimeric molybdenum-containing enzymes (Malasarnet al. (2008), J Bacteriol, 190, 135-142). However, As^(III) oxidasediffers from other molybdenum-containing enzymes in that it is a muchlarger α₂β₂ heterotetramer containing additional co-factors (i.e. a3Fe-4S cluster and a Rieske 2Fe-2S cluster) not present in othermolybdenum-containing enzymes). It has been demonstrated that the nativeenzyme is localised to the periplasm (Santini et al. (2000), JBacteriol, 66, 92-97), given size of the assembled heterotetramericcomplex it is therefore reasonable to speculate that two αβ heterodimersformed in the cytoplasm are exported (via the Tat system) to theperiplasm prior to heterotetrameric complex formation. Therefore, thepresent inventors were surprised to find that that following removal ofthe Tat leader sequence, the modified As^(III) oxidase could achievecomplete assembly (both subunit assembly and cofactor addition) to forma heterotetramer in the cytoplasm of the various E. coli strains.

For the avoidance of doubt, the abbreviations “aio”, “Aio”, “aro”,“Aro”, “aso” and “Aso” can be used interchangeably and all refer to thearsenite oxidase enzyme (gene or protein). The different abbreviationsare the result of different nomenclature that has been used in the art(Lett et al., J. Bacteriology, 4 Nov. 2011; p.207-208).

The advantage of modifying the native As^(III) oxidase according to theinvention is that is can be expressed successfully in heterologousexpression systems such as E. coli at high, commercially-viable yields.Once expressed, the enzyme can be used to detect the presence ofAs^(II). Therefore, a second aspect of the invention is directed to theuse of the modified arsenite oxidase enzyme according to the firstaspect of the invention as a biosensor to detect the presence ofAs^(III) in a sample. The modified enzyme of the invention has beenfound to be equally effective as the native enzyme in catalysing theoxidation of As^(III) to arsenate As^(v) (Warelow & Santini, unpublisheddata). The absence of the translocation sequence does not impact uponthe catalytic activity of the enzyme. The modified enzyme is thereforesuitable for use in biosensors to detect the presence of As^(III).

Preferably the sample is a liquid sample. The liquid sample may be anytype of liquid that is susceptible to As^(III) contamination, including,but not limited to, ground-water, drinking-water, environmental liquidssuch as mining effluent and sewage, waste-water, biological samples.

Preferably the modified enzyme of the invention may be used inlaboratory-based biosensors or, preferably, in low-cost disposablebiosensor suitable for field use (i.e. detecting As^(III) in a sample atthe source of the sample rather than in a laboratory).

The advantage of utilising the modified enzyme according to the firstaspect of the invention in a biosensor for detecting As^(III) in asample is that it can be expressed in commercially-viable quantities ina variety of heterologous expression systems, including, unexpectedly,host strains that are normally used for cloning rather than proteinexpression.

Therefore, according to a third aspect the present invention provides amicroorganism modified to express the heterologous As^(III) oxidase ofthe first aspect of the invention. The wild-type microorganism may beselected from a range of species including, but not limited to, E. coli.Preferably, the wild-type microorganism is E. coli, in particular E.coli K12 strains DH5α and JM109λpir.

It is surprising that these strains have been found to be the mostsuitable for expressing the recombinant enzyme of the invention, sincethey are usually used in the art for cloning, rather than proteinexpression. As detailed in Example 1 below, the inventors found thatunexpectedly high yields were achieved using E. coli strains DH5α andJM109λpir rather than strains BL21 (protease-deficient) and Origami,which are commonly used for heterologous expression of recombinantenzymes.

E. coli strain DH5α is a Hoffman-Berling 1100 strain derivative and canbe purchased from Invitrogen™. The properties of this strain aredescribed by the following standard nomenclature:

-   F-endAl glnV44 thi-1 recAl relAl gyrA96 deoR nupG Φ80dlacZAM15    Δ(lacZYA-argF)U169, hsdR17(rK−mK+), λ−.

The DH5α strain is described in the following publications: FOCUS (1986)8:2, 9.; Hanahan, D. (1985) in DNA Cloning: A Practical Approach(Glover, D. M., ed.), Vol. 1, p. 109, IRL Press; Grant, S. G. N. et al.(1990) Proc. Natl. Acad. Sci. USA 87: 4645-4649 PMID 2162051; andMeselson M. and Yuan R. (1968) Nature 217:1110 PMID. 4868368.

E. coli strain JM109λpir can be purchased from Promega and has thefollowing standard nomenclature properties:

-   endAl glnV44 thi-1 relAl gyrA96 recAl mcrB+ Δ(lac-proAB) e14-[F′    traD36 proAB+ laclq 1acZAM15] hsdR17(rK−mK+).

This strain is described in Yanisch-Perron et al (1985) Gene,33(1):103-19; and Penfold et al (1992) Gene 118:145-146.

According to a fourth embodiment, the present invention provides amethod of producing recombinant As^(III) oxidase, comprising expressingthe modified enzyme according to the first aspect of the invention in aheterologous microorganism. According to an embodiment of this aspect ofthe invention, novel primers identified herein as SEQ ID NO. 9 and SEQID NO. 10 can be used to clone the NT-26 aioB and aioA genes (termedaioBA) into the commercially-available pPROEX Htb vector (Invitrogen).As described in relation to the first aspect of the invention, the aioBgene is modified to delete at least the portion of the nucleotidesequence (SEQ ID NO. 6) encoding at least the functional portion of theTAT leader sequence shown herein as SEQ ID NO. 2.

Preferably the heterologous microorganism is E. coli, and preferably E.coli strains DH5α or JM109λpir. The present inventors have found thatstrain DH5α provides optimal expression of the heterologous arseniteoxidase enzyme when grown aerobically and JM109λpir provides optimalexpression of the heterologous enzyme when grown anaerobically withnitrate as the electron acceptor. Furthermore the inventors have foundthat use of an affinity tag at the N-terminus of AioB allows for simplepurification. This embodiment is described in more detail in Example 1below.

According to a fifth aspect, the present invention provides a device fordetecting the presence of As^(III) in a sample. The device comprises anelectrode (termed the “test electrode”) incorporating an As^(III)oxidase enzyme and a redox mediator.

The device may comprise wild-type As^(III) oxidase or the modifiedAs^(III) oxidase according to the first aspect of the present invention.Preferably, the enzyme is the modified As^(III) oxidase. As discussedabove, high yields of the modified enzyme can be obtained throughheterologous expression in E. coli. Therefore, it is preferable to usethe modified enzyme to keep the manufacturing cost of the device low andto enable the device to be manufactured in commercially-viablequantities.

In one embodiment, the device comprises a test strip made of polymer orceramic materials. Preferably, the device comprises two or more planarelectrodes. Preferably the device comprises a “reference electrode” inaddition to the test electrode. At least the test electrode incorporatesthe As^(III) oxidase.

The device includes a redox mediator. The term “redox mediator” isdefined as any moiety capable of transferring electrons from the enzymeto the electrode surface. Artificial redox mediators such as2,6-dichlorophenolindophenol (DCPIP) are often used in solution inlaboratory-based spectrophotometric measurements; howeverspectrophotometric measurements are not routinely used in field testequipment. The inclusion of a redox mediator is advantageous in a numberof different ways, compared with existing devices in which the enzyme isdirectly linked to an electrode. The presence of a redox mediatorimproves the efficiency of electron transfer and reduces the effects ofelectrode surface chemistry on the currents measured. This enables amuch wider range of electrode materials to be used. As the backgroundcurrents due to sample components are dependent on the electrodematerial this gives greater versatility in optimising signal tobackground.

As shown in Example 2, a wide range of mediators can be used in the inthe electrochemical As^(III)-sensing device of the invention. Examplesof suitable redox mediators include, but are not limited to, metalcomplexes where the metal exists in two or more different redox states,for example iron, ruthenium or osmium complexes, organic molecules thatcan exist in two or more accessible redox states, for examplecytochromes, conducting organic polymers, conducting organic salts,2,6-dichlorophenolindophenol, ferrocene and ferrocene derivatesincluding ferrocene carboxylic acid and hydroxymethyl ferrocene(ferrocene methanol), Tris(2,2′-bipyridine)dichlororuthenium(II),tetrathiafulvalene (TTF) and quinones including, but not limited to,benzoquinone and hydroquinone. However not all redox mediators areeffective with Aio (arsenite oxidase), for example tetraammine ruthenium(III) chloride, methylene blue, ferricyanide and oxygen have been foundnot to be effective.

Advantageously, the device of the invention is versatile and has beenshown to work with a variety of test electrode materials (see Example 2below). Preferably, the test electrode is made of one or more conductingmaterials including, but not limited to, carbon, carbon nanotubes,graphene, graphite, gold, platinum, palladium, glassy carbon,nanostructured metal oxides or nanostructured metal.

Preferably, the reference electrode comprises a reference redox couple,such as Ag/AgCl.

The electrode materials can be deposited on the test strip by a varietymethods including, but not limited to, screen-printing or evaporation.The electrodes may be open or covered by a lid so forming a definedvolume cell. There may, or may not, be a membrane covering theelectrodes.

The test strip is suitable for laboratory use and, preferably,field-based use (i.e. As^(III) can be detected in a sample at the sourceusing the test strip or device). The device may be suitable for multipleuses or a single use, and may be disposable.

Preferably, the device comprises a micro-structured surface, with theenzyme entrapped thereon with a mediator (see FIG. 1). Amicro-structured surface, for example pillars rising from the base ofthe electrode, improves the performance of the electrode.

Preferably the sample in which As^(III) is detected using the device ofthe invention is a liquid sample. The liquid sample may be any type ofliquid that is susceptible to As^(III) contamination, including, but notlimited to, ground-water, drinking-water, environmental liquids such asmining effluent and sewage, waste-water, biological samples.

In use, the test sample is brought into direct contact with the teststrip. Operation of the sensor device involves applying an electricalpotential between the test and reference electrodes and measuring thecurrent. A number of methods would be apparent to those skilled in theart, and include, but are not limited to, chronoamperomerty, square wavevoltammetry and coulometry. The response to As^(III) can be measuredfrom 5 μM to 40 μM with the same sensor showing comparable results thenext day (see FIG. 2).

The advantages of using the device of the invention as described aboveto test for the presence of As^(III) in a sample are that thesensitivity for the analyte is high and the test is specific forAs^(III) (i.e. the most toxic soluble form of arsenic) due to thepresence of the enzyme; the test is quick and results are obtainedalmost instantaneously; the device is simple to use; the results arereproducible; and the device can be produced and purchased cheaply(particularly important for use in developing countries).

The present invention is further described with reference to thefollowing examples.

EXAMPLE 1 Experimental Procedures

Bacterial Strains, Plasmid and Growth Conditions

Escherichia coli strains DH5α, JM109λpir, RK4353, C43, BL21 and Origamiwere used to test expression of the NT-26 arsenite oxidase. Theexpression vector pPROEX-HTb (Invitrogen) was used for expressionpurposes. All expression conditions involved growing E. coli in LBcontaining 100 μg/ml ampicillin either aerobically (with aeration at 170rpm (1:5 ratio liquid to head space) or anaerobically with nitrate (14mM) or DMSO (14 mM) as electron acceptors and sodium lactate (20 mM) asthe electron donor.

Cloning and Expression

The NT-26 aioB and aioA (aioBA) genes were amplified without the nativeTat leader sequence using the following primers:

Forward: PROEXAroBFHis (SEQ ID No. 9)5′-GCGAATTCAAGCTACCGCGGCGGCAGGGGTC-3′ Reverse: PROEXAroAR(SEQ ID No. 10) 5′-GCCTGCAGTCAAGCCGACTGGTATTCTTTCGA-3′

The restriction enzymes EcoRI and Pstl (underlined above) were used tofacilitate cloning into the expression vector, pPROEX-HTb. ThepPROEX-HTb carrying the aioBA genes was transformed into a variety of E.coli strains to determine which one gave optimal expression. A varietyof IPTG (Isopropyl β-D-1-thiogalactopyranoside) and sodium molybdate(Mo) concentrations as well as time of induction were tested. The finaloptimum expression conditions used for purification of the Aio involvedgrowing DH5α aerobically at 21° C. for 24 hrs in LB containing 40 μMIPTG and 1 mM Mo.

Purification of Recombinant Arsenite Oxidase

Recombinant Aio was purified from DH5α using a combination of affinityand size exclusion chromatography. Cells were harvested bycentrifugation at 9,700×g for 10 minutes. The cell pellets were pooledand washed by suspending in binding buffer (10 ml/gm wet weight cells)(20 mM potassium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH7.2) and centrifuged at 12,000×g for 15 minutes. The cell pellet wasthen re-suspended in binding buffer (10 ml/gm wet weight cells). The E.coli cells were disrupted by a single pass through a French pressurecell (12,000 psi) and the cell debris removed by centrifugation at30,000×g for 30 minutes. The supernatant was loaded onto a GraviTrappre-packed Ni charged affinity chromatography column (GE Healthcare) asper the manufacturer's instructions except with one minor modification;the wash volume used was 120 ml. The eluent was desalted in 50 mM2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) buffer resulting inthe precipitation of non-target co-eluted protein(s) which were removedby centrifugation at 10,000×g for 5 minutes. The supernatant wasfiltered through a 0.22 μm filter (Millipore), concentrated using aVivaspin 20 (MWCO 100,000) (GE Healthcare) centrifugal concentrator andloaded onto a Superdex 200 gel filtration column (GE Healthcare)pre-equilibrated with 50 mM MES, 100 mM NaCl, pH 5.5 buffer.Chromatography was carried out at a flow rate of 0.3 ml/min. The 0.25 mlfractions containing Aio activity were pooled and concentrated using aVivaspin 20 centrifugal concentrator (MWCO 100,000).

Confirmation of the native molecular mass of the recombinant Aio wasdone using a Superdex 200 gel filtration (GE Healthcare) chromatographycolumn. A calibration curve was created using a gel filtrationcalibration kit (GE Healthcare) of known molecular mass proteins(Thyroglobulin 669 kDa, Ferritin 440 kDa, Aldolase 158 kDa, Conalbumin75 kDa, Ovalbumin 43 kDa) and the void volume of the column wasdetermined using Blue dextran 2000 (2,000 kDa). Chromatographyconditions used were as per the manufacturers' instructions with a flowrate of 0.3 ml/min.

Results and Discussion Heterologous Expression and Purification of theRecombinant Arsenite Oxidase

In this study the aioB and aioA (designated aioBA) genes were clonedwithout the aioB Tat leader sequence allowing for expression in the E.coli cytoplasm. A combination of different strains and growth conditionswere tested to optimise Aio expression. Surprisingly, the highest Aiospecific activity was obtained with E. coli K12 strains DH5α andJM109λpir.

Interestingly, there was also a variation of Aio activity when differentelectron acceptors were used (FIG. 3), with nitrate the optimal electronacceptor. Given the greater cell yield obtained from aerobic growth,DH5α and oxic conditions were chosen for further studies. RecombinantAs^(III) oxdase was purified from E. coli using a combination of Ni-NTAand size exclusion chromatography. Based on SDS polyacrylamide gelelectrophoresis, the recombinant enzyme was >99% pure and contained thetwo known Aio subunits, AioA (93 kDa) and AioB (21 kDa) (see FIG. 4).Based on size exclusion chromatography the native molecular mass of theenzyme was 219 kDa which is consistent with the α₂β₂ oligomeric state ofthe native enzyme purified from NT-26 (Santini & vanden Hoven, 2004, J.Bacteriology. 186(6):1614-1619).

EXAMPLE 2

A number of experiments were performed to demonstrate that the enzymedescribed within the patent application can be used in an enzymemediated electrochemical device to detect arsenite in water. The resultsdemonstrate that a range of redox mediator molecules can be used in thedevice and that the electrode material can also be varied.

Materials & Methods

Screen printed electrodes were purchased from Dropsens (Oviedo, Spain).Carbon screen printed electrodes have a carbon working electrode (4mmdiameter) and counter electrode and silver/silver chloride referenceelectrode (product ref. DRP-C 110). Carbon nanotube screen printedelectrodes have a multi-walled carbon nanotube working electrode (4 mmdiameter), carbon counter electrode and silver/silver chloride referenceelectrode (produce reference DRP-110CNT). Gold screen printed electrodehave a gold working electrode (4 mm diameter) and counter electrode anda silver/silver chloride reference electrode (product referenceDRP-220AT). Electrochemical measurements (cyclic voltammetry and chronoamperometry) were recorded using a CompactStat.e potentiostat instrument(Ivium Technologies) and IviumSoft software (Ivium Technologies).2,6-dichlorophenolindophenol, Ferrocene Carboxylic acid, hydroxymethylferrocene (ferrocene methanol), tetrathiafulvalene (TTF) andTris(2,2′-bipyridine)dichlororuthenium(II) mediators were all purchasedfrom Sigma Aldrich. 50 mM solution of sodium arsenite (Fluka) was alsopurchased from Sigma Aldrich.

In all experiments the working, counter and reference electrodes werecompletely covered with a 200 μl buffered solution containing therequired amount of mediator, arsenite oxidase enzyme and arsenite foreach experiment.

Cyclic voltammograms were recorded using carbon, carbon nanotube andgold electodes with a 0.5 mM hydroxymethyl ferrocene solution (10 mMPBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17nanomoles) with and without 500 micromolar sodium arsenite. A scan rateof 5 mV/s was used. Chrono amperometry was performed using a 0.25 mMhydroxymethyl ferrocene solution (10 mM PBS, pH 7.1, 100 mM KCl)containing 0.05 U arsenite oxidase (0.17 nanomoles) with 0 and 50micromolar sodium arsenite when using a carbon electrode and 0, 12.5,25, 50 and 100 micromolar sodium arsenite when using a gold electrode.The solution was mixed between each chrono amperometry run. Triplicateruns were recorded for each concentration of arsenite and the averagevalues plotted. A one step potential of 0.35 V (vs Ag/AgCl) was appliedfor each run.

Cyclic voltammograms were recorded using carbon and carbon nanotubeelectodes with a 1 mM Tris(2,2′-bipyridine)dichlororuthenium(II)solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.05 U arseniteoxidase (0.17 nanomoles) with and without 500 micromolar sodiumarsenite. A scan rate of 5 mV/s was used.

Cyclic voltammograms were recorded using carbon and gold electodes witha 1 mM ferrocene carboxylic acid solution (10 mM PBS, pH 7.1, 100 mMKCl) containing 0.1 U arsenite oxidase (0.34 nanomoles) with and without1 mM sodium arsenite. A scan rate of 5 mV/s was used. Chrono amperometrywas performed using a 1 mM ferrocene carboxylic acid solution (10 mMPBS, pH 7.1, 100 mM KCl) containing 0.1 U arsenite oxidase (0.34nanomoles) with 0, 50 and 100 micromolar sodium arsenite with a carbonelectrode. A single step run was performed applying a potential of 0.38V (vs Ag/AgCl reference electrode). The solution was mixed between eachchrono amperometry run. Duplicate runs were recorded for eachconcentration of arsenite.

Cyclic voltammograms were recorded using carbon, carbon nanotube andgold electodes with a 1 mM 2,6-dichlorophenolindophenol (DCPIP) solution(50 mM MES, pH 5.5, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17nanomoles) with and without 500 micromolar sodium arsenite. A scan rateof 5 mV/s was used.

Cyclic voltammograms were recorded using carbon nanotube electode byfirst depositing 6 μl of 2 mM tetrathiafulvalene (TTF) in acetone ontothe working electrode and allowing to dry for 10 minutes. The electrodescreen printed electrode was then immersed in 200 μl solution (10 mMPBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17nanomoles) with and without 500 micromolar sodium arsenite. A scan rateof 50mV/s was used.

Results

2,6-dichlorophenolindophenol, Ferrocene Carboxylic acid, FerroceneMethanol and Tris(2,2′-bipyridine)dichlororuthenium(II) mediators allshowed reversible redox behaviour in the presence of the enzyme (verysimilar cathodic and anodic peak currents) and an increased cathodicpeak current relative to the anodic peak current in the presence of theenzyme and arsenite. This is a typical response of an enzyme mediatedelectrochemical device.

FIGS. 5(a-f) show cyclic voltammograms of ferrocene methanol as themediator with carbon (a and b), carbon nanotube (c and d) and gold (eand f) screen printed electrodes without (a, c, e) and with 500micromolar arsenite (b,d,f). FIG. 5g shows chronoamperometry plots offerrocene methanol plus enzyme, and ferrocene methanol plus enzyme plus50 μM arsenite recorded using a carbon electrode. FIGS. 5h and 5i showchronoamperometry plots of ferrocene methanol plus enzyme, and ferrocenemethanol plus enzyme plus 0, 12.5, 25, 50 and 100 micromolar arsentierecorded using a gold electrode. FIG. 5i is a zoomed portion of FIG. 5h. FIG. 5j is a plot showing a linear dose response of current toarsenite concentration and refers to the Chrono amperometry plots inFIGS. 5h and 5i , plotting current values taken at four seconds from thechrono amperometry plots.

FIGS. 6(a-d) show cyclic voltammograms of Tris(2,2′-bipyridine)dichlororuthenium(II) as the mediator with carbon (a and b) and carbonnanotube (c and d) screen printed electrodes, without (a and c) and with(b and d) 500 micromolar arsenite.

FIGS. 7a and 7b show cyclic voltammograms of feccocene carboxylic acidas the mediator with carbon (2a) and gold (2b) screen printedelectrodes. FIG. 7c shows chrono amperometry plots of ferrocenecarboxylic acid plus enzyme with 0, 50 micromolar and 100 micromolararsenite.

FIGS. 8(a-f) show cyclic voltammograms of 2,6-dichlorophenolindophenolas the mediator with carbon (a and b), gold (c and d) and carbonnanotube (e and f) screen printed electrodes, without (a,c,e) and with(b,d,f) 500 micromolar arsenite.

FIG. 9 shows cyclic voltammograms of TTF as the mediator with carbonnanotube screen printed electrodes, without and with 500 micromolararsenite.

Chronoamperometry plots show a shift to high current values in thepresence of arsenite and there was a linear dose response in the currentshift to the arsenite concentration.

1. An arsenite oxidase enzyme modified to prevent translocation to theperiplasm, wherein the enzyme comprises the native AioA subunit fromNT-26, or a variant, homologue or derivative thereof, and the nativeAioB subunit from NT-26, or a variant, homologue or derivative thereof,wherein a portion of the native AioB subunit corresponding to thetranslocation signal sequence, or a functional fragment thereof, ismodified.
 2. A modified enzyme according to claim 1, wherein a portionof the native aioB gene which encodes the translocation signal sequence,or a portion thereof encoding a functional fragment of the translocationsignal sequence, is modified.
 3. A modified enzyme according to claim 2,wherein the portion of the native aioB gene is identified as SEQ ID NO.6, or a homologue of SEQ ID NO. 6 encoding a functional fragment of thetranslocation signal sequence identified as SEQ ID NO.
 2. 4. A modifiedenzyme according to claim 1, wherein the modification is deletion.
 5. Amodified enzyme according to claim 1, wherein the AioB subunit comprisesof the peptide sequence identified a SEQ ID NO. 3, or a variant,homologue or derivative thereof, or is encoded by the polynucleotidesequence of SEQ ID NO. 7, or a variant, homologue or derivative thereof.6. A modified enzyme according to claim 1, wherein the AioA subunitcomprises the peptide sequence identified as SEQ ID NO. 4, or a variant,homologue or derivative thereof, or is encoded by the polynucleotidesequence of SEQ ID NO. 8, or a variant, homologue or derivative thereof.7. Use of a modified arsenite oxidase enzyme according to claim 1 in abiosensor to detect the presence of arsenite in a sample.
 8. Useaccording to claim 7, wherein the sample is a liquid sample.
 9. Amicroorganism modified to express a heterologous arsenite oxidase enzymeas defined in claim
 1. 10. A modified microorganism according to claim9, wherein the microorganism is E. coli.
 11. A modified microorganismaccording to claim 10, wherein the microorganism is E. coli strain isDH5a or JM109λpir.
 12. A method of producing recombinant arseniteoxidase, comprising expressing the modified arsenite oxidase enzymeaccording to claim 1 in a heterologous microorganism.
 13. A methodaccording to claim 12, wherein the heterologous microorganism is E.coli.
 14. A method according to claim 13, wherein the E. coli. strain isDH5a or JM109λpir.
 15. A method according to claim 12, comprisingcloning modified aioBA into a vector, wherein the portion of the nativeaioB gene which encodes the translocation signal sequence, or a portionthereof encoding a functional fragment of the translocation signalsequence, is modified.
 16. A method according to claim 15, wherein thevector is the pPROEX HTb vector.
 17. A method according to claim 15,wherein the primer sequences identified herein as SEQ ID NO. 9 and/orSEQ ID NO. 10 are to clone the modified genes into the vector.